Dennis Kappei

This is an important example but the responsibility is not only with vendors - journals need to systematically require antibody validation: antibody off-targets may vary in rel. expression levels, i.e. an antibody that appears "clean" in cell line A might be problematic in cell line B.

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In a nutshell, we propose that ZBTB48 is a priming factor that opens up its (few) target genes to then give access for other TFs. In such a model, ZBTB48 acts as an on-off-switch while other TFs fine-tune how much mRNA is produced.

Finally, we wanted to understand how ZBTB48 regulates CIITA (and other genes). In line with transcriptional regulation the H3K4me3 signal at CIITA pIII was entirely dependent on ZBTB48 and upstream of this ZBTB48 was also required for chromatin access as assayed by FAIREseq.

Does this matter at the organismal level? We created a Zbtb48 KO strain and observed that MHC II surface levels in B cells were also strongly reduced, with the most striking effect in pre-B cells. Interestingly, female (but not male) KO mice presented with mild splenomegaly.

To test a functional impact, we induced CIITA/MHCII expression by IFNgamma treatment. In contrast to WT cells, ZBTB48 KO cells had strongly reduced CIITA & MHCII mRNA and protein levels. The CIITA pIII specific transcript was almost entirely shut off.

We biochemically mapped the binding sites and ZBTB48 binds directly to the two critical activating elements, ARE1/2. A corresponding co-crystal structure revealed involvement of ZnF10 & ZnF11 with the former dispensable for ZBTB48's telomeric function --> separation-of-function.

While revisiting prior ChIP-seq data we realised that we had overlooked a particularly exciting target gene, CIITA, because it is not expressed in non-immune cells. CIITA expression depends on 3 promoters and ZBTB48 sits right on top of the B-cell-specific promoter pIII.

ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator | EMBO reports image image The zinc finger protein ZBTB48 directly binds telomeres and limits their elongation. ZBTB48 also transcriptionally activates a defined set of genes, including mitochondrial fission process 1, linking telomere length regulation ...

We had previously identified ZBTB48 as a telomere length regulator moonlighting as a transcriptional activator (embopress.org/doi/full/10....). While we knew that ZBTB48 was an unusual TF - huge effects on a small number of genes - we wanted to understand how it actually functioned.

So wonderful to see our recent work on the cover of
@embojournal.bsky.social, beautifully illustrated by
@drawimpacts.bsky.social
- not only visualising our scientific discovery but also celebrating our beautiful environment in #Singapore. 🤩